Formation of Supplementary Metal-Binding Centers in Proteins under Stress Conditions.

In many proteins, supplementary metal-binding centers appear under stress conditions. They are known as aberrant or atypical sites. Physico-chemical properties of proteins are significantly changed after such metal binding, and very stable protein aggregates are formed, in which metals act as "cross-linking" agents. Supplementary metal-binding centers in proteins often arise as a result of posttranslational modifications caused by reactive oxygen and nitrogen species and reactive carbonyl compounds. New chemical groups formed as a result of these modifications can act as ligands for binding metal ions. Special attention is paid to the role of cysteine SH-groups in the formation of supplementary metal-binding centers, since these groups are the main target for the action of reactive species. Supplementary metal binding centers may also appear due to unmasking of amino acid residues when protein conformation changing. Appearance of such centers is usually considered as a pathological process. Such unilateral approach does not allow to obtain an integral view of the phenomenon, ignoring cases when formation of metal complexes with altered proteins is a way to adjust protein properties, activity, and stability under the changed redox conditions. The role of metals in protein aggregation is being studied actively, since it leads to formation of non-membranous organelles, liquid condensates, and solid conglomerates. Some proteins found in such aggregates are typical for various diseases, such as Alzheimer's and Huntington's diseases, amyotrophic lateral sclerosis, and some types of cancer.

Antiglycation and Antioxidant Effect of Nitroxyl towards Hemoglobin.

Donors of nitroxyl and nitroxyl anion (HNO/NO-) are considered to be promising pharmacological treatments with a wide range of applications. Remarkable chemical properties allow nitroxyl to function as a classic antioxidant. We assume that HNO/NO- can level down the non-enzymatic glycation of biomolecules. Since erythrocyte hemoglobin (Hb) is highly susceptible to non-enzymatic glycation, we studied the effect of a nitroxyl donor, Angeli's salt, on Hb modification with methylglyoxal (MG) and organic peroxide-tert-butyl hydroperoxide (t-BOOH). Nitroxyl dose-dependently decreased the amount of protein carbonyls and advanced glycation end products (AGEs) that were formed in the case of Hb incubation with MG. Likewise, nitroxyl effectively protected Hb against oxidative modification with t-BOOH. It slowed down the destruction of heme, formation of carbonyl derivatives and inter-subunit cross-linking. The protective effect of nitroxyl on Hb in this system is primarily associated with nitrosylation of oxidized Hb and reduction of its ferryl form, which lowers the yield of free radical products. We suppose that the dual (antioxidant and antiglycation) effect of nitroxyl makes its application possible as part of an additional treatment strategy for oxidative and carbonyl stress-associated diseases.

Protective Effect of Dinitrosyl Iron Complexes Bound with Hemoglobin on Oxidative Modification by Peroxynitrite.

Dinitrosyl iron complexes (DNICs) are a physiological form of nitric oxide (•NO) in an organism. They are able not only to deposit and transport •NO, but are also to act as antioxidant and antiradical agents. However, the mechanics of hemoglobin-bound DNICs (Hb-DNICs) protecting Hb against peroxynitrite-caused, mediated oxidative modification have not yet been scrutinized. Through EPR spectroscopy we show that Hb-DNICs are destroyed under the peroxynitrite action in a dose-dependent manner. At the same time, DNICs inhibit the oxidation of tryptophan and tyrosine residues and formation of carbonyl derivatives. They also prevent the formation of covalent crosslinks between Hb subunits and degradation of a heme group. These effects can arise from the oxoferryl heme form being reduced, and they can be connected with the ability of DNICs to directly intercept peroxynitrite and free radicals, which emerge due to its homolysis. These data show that DNICs may ensure protection from myocardial ischemia.

Carbonyl Stress in Red Blood Cells and Hemoglobin.

The paper overviews the peculiarities of carbonyl stress in nucleus-free mammal red blood cells (RBCs). Some functional features of RBCs make them exceptionally susceptible to reactive carbonyl compounds (RCC) from both blood plasma and the intracellular environment. In the first case, these compounds arise from the increased concentrations of glucose or ketone bodies in blood plasma, and in the second-from a misbalance in the glycolysis regulation. RBCs are normally exposed to RCC-methylglyoxal (MG), triglycerides-in blood plasma of diabetes patients. MG modifies lipoproteins and membrane proteins of RBCs and endothelial cells both on its own and with reactive oxygen species (ROS). Together, these phenomena may lead to arterial hypertension, atherosclerosis, hemolytic anemia, vascular occlusion, local ischemia, and hypercoagulation phenotype formation. ROS, reactive nitrogen species (RNS), and RCC might also damage hemoglobin (Hb), the most common protein in the RBC cytoplasm. It was Hb with which non-enzymatic glycation was first shown in living systems under physiological conditions. Glycated HbA1c is used as a very reliable and useful diagnostic marker. Studying the impacts of MG, ROS, and RNS on the physiological state of RBCs and Hb is of undisputed importance for basic and applied science.

XANES Measurements for Studies of Adsorbed Protein Layers at Liquid Interfaces.

X-ray absorption near edge structure (XANES) spectra for protein layers adsorbed at liquid interfaces in a Langmuir trough have been recorded for the first time. We studied the parkin protein (so-called E3 ubiquitin ligase), which plays an important role in pathogenesis of Parkinson disease. Parkin contains eight Zn binding sites, consisting of cysteine and histidine residues in a tetracoordinated geometry. Zn K-edge XANES spectra were collected in the following two series: under mild radiation condition of measurements (short exposition time) and with high X-ray radiation load. XANES fingerprint analysis was applied to obtain information on ligand environments around zinc ions. Two types of zinc coordination geometry were identified depending on X-ray radiation load. We found that, under mild conditions, local zinc environment in our parkin preparations was very similar to that identified in hemoglobin, treated with a solution of ZnCl2 salt. Under high X-ray radiation load, considerable changes in the zinc site structure were observed; local zinc environment appeared to be almost identical to that defined in Zn-containing enzyme alkaline phosphatase. The formation of a similar metal site in unrelated protein molecules, observed in our experiments, highlights the significance of metal binding templates as essential structural modules in protein macromolecules.

Natural killer cell cytotoxic activity and c-Fos protein synthesis in rat hypothalamic cells after painful electric stimulation of the hind limbs and EHF irradiation of the skin.

BACKGROUND: The goal was to assess changes in natural killer (NK) cell activity and the number of c-Fos-positive cells in hypothalamic structures induced by painful electrical stimulation and to use extremely high-frequency (EHF) irradiation of the skin to modulate these processes. MATERIAL/METHODS: Experiments were performed on Wistar rats subjected to painful electrical stimulation of the hind limbs combined with EHF irradiation of the skin. The cytotoxic activity of splenic NK cells was assessed by their ability to lyse K-562 tumor cells in vitro. c-Fos-like protein was detected by an immunoperoxidase technique. RESULTS: Painful electric stimulation was associated with a significant decrease in splenic NK cytotoxicity and a dramatic increase in c-Fos-positive cell counts in some hypothalamic structures, particularly in the anterior hypothalamic nucleus (AHN) and the perifornical lateral hypothalamic area (LHA). Two EHF exposures, one before and one after electric stimulation, prevented the suppression of splenic NK cell activity and caused a decrease in the number of c-Fos-positive cells expressed in the ventromedial hypothalamic nucleus (VMH) and basal LHA. Negative correlation was shown between c-Fos-positive cell counts in the AHN and LHA and the cytotoxic activity of NK cells. CONCLUSIONS: These results suggest that painful electric stimulation of the hind limbs of rats causes a reorganization of the central mechanisms that regulate splenic NK cell activity, resulting in a decrease in their cytotoxicity, and that EHF irradiation of the skin prevents this reorganization, thus protecting splenic NK cell activity from the impairment induced by this stressor.

X-ray fluorescence methods for investigations of lipid/protein membrane models.

The protective effect of the bisphosphonate drug xydiphone (K,Na-ethidronate) on membrane-bound enzyme damaged by lead ions has been studied. A protein/lipid film of Ca-ATPase/phosphatedylethanolamine deposited on a silicon substrate was used as a model system. The position of lead ions within the molecular film before and after the xydiphone treatment was determined using the total-reflection X-ray fluorescence method. This technique is based on the simultaneous measurement of the X-ray reflection and the yield of the fluorescence radiation excited by X-ray inelastic scattering. The possibility of directly locating lead ions is the main advantage of this approach. Xydiphone has been found to effectively eliminate lead ions that have been incorporated into Ca-ATPase molecules during a preliminary incubation in lead acetate solution. The lead ions that were bound at the sites of the Ca-ATPase attachment to the phospholipid monolayer have proved to be inaccessible for xydiphone. A preliminary incubation of Ca-ATPase in the xydiphone solution precluded the incorporation of lead ions into the protein.